Recombinant Multi-l-Arginyl-Poly-l-Aspartate (Cyanophycin) as an Emerging Biomaterial.

Multi-l-arginyl-poly-l-aspartate (MAPA) is a non-ribosomal polypeptide which synthesis is directed by cyanophycin synthetase, and its production can be achieved using recombinant microorganisms carrying the cphA gene. Along its poly-aspartate backbone, arginine or lysine links to each aspartate via an isopeptide bond. MAPA is a zwitterionic polyelectrolyte full of charged carboxylic, amine, and guanidino groups. In aqueous solution, MAPA exhibits dual thermal and pH responses similar to those stimuli-responsive polymers. Being biocompatible, the films containing MAPA can support cell proliferation and elicits minimal immune response in macrophages. Dipeptides from MAPA after enzymatic treatments can provide nutritional benefits. In light of the increasing interest in MAPA, this article focuses on the recent discovery of the function of cyanophycin synthetase and the potentials of MAPA as a biomaterial.

Altering the substrate specificity of recombinant l-rhamnose isomerase from Thermoanaerobacterium saccharolyticum NTOU1 to favor d-allose production.

l-Rhamnose isomerase (l-RhI) catalyzes rare sugar isomerization between aldoses and ketoses. In an attempt to alter the substrate specificity of Thermoanaerobacterium saccharolyticus NTOU1 l-RhI (TsRhI), residue Ile102 was changed to other polar or charged amino acid residues by site-directed mutagenesis. The results of activity-screening using different substrates indicate that I102N, I102Q, and I102R TsRhIs can increase the preference against d-allose in comparison with the wild-type enzyme. The catalytic efficiencies of the purified I102N, I102Q, and I102R TsRhIs against d-allose are 148 %, 277 %, and 191 %, respectively, of that of wild-type enzyme, while those against l-rhamnose are 100 %, 167 % and 87 %, respectively. Mutant I102N, I102Q, and I102R TsRhIs were noted to have the altered substrate specificity, and I102Q TsRhI has the highest catalytic efficiency against d-allose presumably through the formation of an additional hydrogen bond with d-allose. The purified wild-type and mutant TsRhIs were further used to produce d-allose from 100 g/L d-fructose in the presence of d-allulose 3-epimerase, and the yields can reach as high as 22 % d-allulose and 12 % d-allose upon equilibrium. I102Q TsRhI takes only around half of the time to reach the same 12 % d-allose yield, suggesting that this mutant enzyme has a potential to be applied in d-allose production.

Wound Healing Attributes of Polyelectrolyte Multilayers Prepared with Multi-l-arginyl-poly-l-aspartate Pairing with Hyaluronic Acid and γ-Polyglutamic Acid.

Biodegradable multi-l-arginyl-poly-l-aspartate (MAPA), more commonly cyanophycin, prepared with recombinant Escherichia coli contains a polyaspartate backbone with lysine and arginine as side chains. Two assemblies of polyelectrolyte multilayers (PEMs) are fabricated at three different concentration ratios of insoluble MAPA (iMAPA) with hyaluronic acid (iMAPA/HA) and with γ-polyglutamic acid (iMAPA/γ-PGA), respectively, utilizing a layer-by-layer approach. Both films with iMAPA and its counterpart, HA or γ-PGA, as the terminal layer are prepared to assess the effect on film roughness, cell growth, and cell migration. iMAPA incorporation is higher for a higher concentration of the anionic polymer due to better charge interaction. The iMAPA/HA films when compared to iMAPA/γ-PGA multilayers show least roughness. The growth rates of L929 fibroblast cells on the PEMs are similar to those on glass substrate, with no supplementary effect of the terminal layer. However, the migration rates of L929 cells increase for all PEMs. γ-PGA incorporated films impart 50% enhancement to the cell migration after 12 h of culture as compared to the untreated glass, and the smooth films containing HA display a maximum 82% improvement. The results present the use of iMAPA to construct a new layer-by-layer system of polyelectrolyte biopolymers with a potential application in wound dressing.

Reversible Self-Assembly Nanovesicle of UCST Response Prepared with Multi-l-arginyl-poly-l-aspartate Conjugated with Polyethylene Glycol.

Multi-L-arginyl-poly-L-aspartate (MAPA), also known as cyanophycin, containing a backbone of polyaspartate with arginine and lysine as side chains, was prepared with recombinant Escherichia coli. The insoluble part (iMAPA) was conjugated with polyethylene glycol (PEG) at two different levels, high (iMAPA(PEG)h) and low (iMAPA(PEG)l). Both levels of conjugation exhibited UCST (upper critical solution temperature)-type responses in the pH range of 3-10 at a concentration of 2 mg/mL. The cloud-point temperature of each conjugate also showed a positive correlation with concentration in PBS, falling between 20 to 58 °C at a concentration from 0.1 to 3 mg/mL. Hysteresis was observed to follow approximate paths under the same condition during repeated heating and cooling. Notably, the reversible formation of core-shell vesicles appeared at room temperature in PBS with a size of around 25 to 60 nm, as measured by DLS and observed under TEM. The reversibility was further employed to encapsulate doxorubicin (Dox) at different weight ratios of Dox to iMAPA(PEG)h. An encapsulation efficiency could reach as high as 70% with an equivalent loading capacity of 1.5 mg Dox/mg iMAPA(PEG)h. The Dox-loaded vesicles stayed stable at 4 °C for up to 4 weeks, with a minimal leakage below 2% and a slightly dilated morphology. Temperature-triggered release of Dox from the vesicles could be achieved by a step change of 5 °C successively from 37 to 62 °C in an effort to induce an initial 10% release at 37 °C gradually to complete release at 62 °C.

Characterization of a recombinant d-allulose 3-epimerase from Agrobacterium sp. ATCC 31749 and identification of an important interfacial residue.

d-Allulose 3-epimerase (DAEase) catalyzes the epimerization between d-fructose and d-allulose. We had PCR-cloned and overexpressed the gene encoding Agrobacterium sp. ATCC 31749 DAEase (AsDAEase) in Escherichia coli. A high yield of active AsDAEase, 35,300U/L or 1350U/g of wet cells, was acquired with isopropyl ß-d-1-thiogalactopyranoside induction at 20°C for 20h. Although only six residues including residue 234 located in tetrameric interface are different between AsDAEase and A. tumefaciens DAEase (AtDAEase), the specific activity of purified AsDAEase is much larger than that of AtDAEase. The optimal pHs and optimal temperatures of the purified recombinant AsDAEase are 7.5-8.0 and 55-60°C, respectively. The half-life of the enzyme is 267min at 55°C in the presence of 0.1mM Co2+, and the equilibrium ratio between d-allulose and d-fructose is 30:70 at 55°C. Besides characterizing AsDAEase, mutation N234D was constructed to assess its influence on activity. The specific activity of the purified N234D AsDAEase is only 25.5% of wild-type's activity, suggesting residue N234 is an important interfacial residue which substantially affects enzyme activity. The high specific activity and high expression yield of AsDAEase suggest its prospect to be applied in d-allulose production.

Overexpression and characterization of a recombinant l-ribose isomerase from Actinotalea fermentans ATCC 43279.

A putative l-ribose isomerase (EC 5.3.1.B3, l-RI) gene of Actinotalea fermentans ATCC 43279 was chemically synthesized, subcloned into pET-21b vector, and then overexpressed in Escherichia coli. After 0.5mM IPTG induction at 20°C for 20h, the recombinant l-RI was highly expressed with up to 50% of the total proteins. About 70% of the expressed l-RI appeared in the cell-free extract as a soluble form, and a high yield of active l-RI, 23,800U/L or 952U/g of wet cells, was achieved. The purified recombinant l-RI demonstrated its optimal activity at 45°C and pH 8 (in tricine-NaOH buffer). Metal ions are not required for l-RI activity, but Hg2+ inhibits its activity completely. The enzyme has a half-life of 74min at 50°C and an equilibrium ratio of 30:70 between l-ribulose and l-ribose at 45°C. The Vmax, kcat, KM, and catalytic efficiency (kcat/KM) of the recombinant l-RI against l-ribose are 232U/mg, 6700min-1, 31.3mM, and 214min-1mM-1, respectively. The high expression yield of the active recombinant A. fermentansl-RI and its highest Vmax, kcat, and catalytic efficiency among the characterized recombinant l-RIs suggest that this recombinant enzyme shows a potential application to produce l-ribose in industry.

Solubility and thermal response of fractionated cyanophycin prepared with recombinant Escherichia coli.

Cyanophycin, also known as cyanophycin granule polypeptide (CGP), is a non-ribosomal polypeptide consisting of aspartic acid as a backbone with arginine and lysine as the side chains. CGP has soluble (sCGP) and insoluble (iCGP) forms based on its aqueous solubility. In order to investigate the role of lysine in its physical properties, CGP was prepared with recombinant Escherichia coli cultivated at different temperatures, and the purified sCGP and iCGP were further fractionated with different ethanol concentrations and pHs, respectively. Low temperature cultivation was found to favor the production of more sCGP. The ratio of iCGP/sCGP increased from 0.12 to 0.35 when the cultivation temperature being raised from 17 to 37°C. After fractionation the same fraction of either sCGP or iCGP contained an approximate content of lysine, and therefore showed expectantly similar physical properties, irrespective of the cultivation temperatures. A high arginine/lysine ratio was found to result in low solubility and high molecular weight. Fractions of sCGP showed upper critical solution temperatures (UCST) below 5°C in phosphate buffered saline whereas iCGP exhibited a UCST around 28-31°C at pH 3. The particle size of iCGP was around 300-500nm as revealed by transmission electron microscopy. The thermal responses of CGP present a potential in biomedical applications, such as drug delivery.

Identification of substrate-binding and selectivity-related residues of maltooligosyltrehalose synthase from the thermophilic archaeon Sulfolobus solfataricus ATCC 35092.

Maltooligosyltrehalose synthase (MTSase) is a key enzyme in the synthesis of trehalose. Computer simulations using AutoDock and NAMD were employed to assess the substrate-binding and selectivity-related residues of MTSase. We introduced mutations at residues D411, D610, and R614 to determine the substrate-binding residues of Sulfolobus solfataricus ATCC 35092 MTSase, and introduced mutations at residues P402, A406, and V426 to investigate the enzyme's selectivity-related residues. Kinetic studies of D411A, D610A, and R614A MTSases reveal significant reductions in catalytic efficiency and cause increase in the transition-state energy of mutant MTSases, indicating that residues D411, D610, and R614 form hydrogen bonds to the substrate. Compared with wild-type MTSase, the hydrolysis: transglycosylation selectivity ratio was significantly decreased for P402Q and significantly increased for A406S MTSases, while the ratio for V426T MTSase showed little change. The results suggest that P402 and A406 residues are selectivity-related.

Interaction between trehalose and MTHase from Sulfolobus solfataricus studied by theoretical computation and site-directed mutagenesis.

Maltooligosyltrehalose trehalohydrolase (MTHase) catalyzes the release of trehalose by cleaving the α-1,4-glucosidic linkage next to the α-1,1-linked terminal disaccharide of maltooligosyltrehalose. Computer simulation using the hydrogen bond analysis, free energy decomposition, and computational alanine scanning were employed to investigate the interaction between maltooligosyltrehalose and the enzyme. The same residues that were chosen for theoretical investigation were also studied by site-directed mutagenesis and enzyme kinetic analysis. The importance of residues determined either experimentally or computed theoretically were in good accord with each other. It was found that residues Y155, D156, and W218 of subsites -2 and -3 of the enzyme might play an important role in interacting with the ligand. The theoretically constructed structure of the enzyme-ligand complex was further validated through an ab initio quantum chemical calculation using the Gaussian09 package. The activation energy computed from this latter study was very similar to those reported in literatures for the same type of hydrolysis reactions.

pH responsive PEGylation through metal affinity for gene delivery mediated by histidine-grafted polyethylenimine.

Polyethylene glycol (PEG) has been used to enhance the stability of a gene delivery system. The most commonly used approach is to add the PEG molecule by way of chemical conjugation. In this study, we prepared PEG-bearing nitrilotriacetic acid (ntaPEG) followed by chelation with either nickel or zinc ions. Polyethylenimine was grafted with histidine (hisPEI) and used as a primary gene carrier to form complexes with DNA. PEGylation was performed by incubating the complexes with chelated ntaPEG. It was noted that the coating of the chelated ntaPEG could provide a shielding effect against aggregation induced by bovine serum albumin and DNA release induced by heparin displacement, respectively. The coating was also found to improve cellular viability and maintain the transfection efficiency at a moderate level. The coated ntaPEG could dissociate from the complexes in an acidic condition of pH 4, suggesting that dePEGylation might occur in some acidic intracellular organelles, such as endosomes. This simple and effective PEGylation approach could be extended to other delivery systems to enhance the stability and to facilitate the dePEGylation process.

Assessments of growth conditions on the production of cyanophycin by recombinant Escherichia coli strains expressing cyanophycin synthetase gene.

The synthesis of cyanophycin, a biodegradable polymer, is directed by cyanophycin synthetase. Polymerase chain reaction (PCR) cloned the gene cphA coding for cyanophycin synthetase from Synechocystis sp. PCC 6803 into pET-21b followed by transformation into two Escherichia coli hosts. The culture conditions for cyanophycin production were investigated, and the molecular weight and compositions of purified cyanophycin were analyzed. The results showed that E. coli BL21-CodonPlus(DE3)-RIL could produce 120 mg cyanophycin per gram dry cell weight in terrific medium. The purified cyanophycin consisted of insoluble and soluble forms at pH 7. The insoluble form had a higher molecular weight (20-32 kDa) than the soluble form (14-25 kDa). Both forms are composed of three major amino acids, aspartic acid, arginine, and lysine, and the insoluble form showed a higher arginine/lysine molar ratio (4.61 ± 0.31) than the soluble form (0.89 ± 0.05). In addition, the nitrogen sources could affect the yields of insoluble and soluble forms of cyanophycin. The medium containing additional lysine could enhance the proportion of the soluble form, but had little effect on the lysine and arginine percentages of both soluble and insoluble forms. The medium containing additional arginine slightly decreased the proportion of soluble form and altered its amino acid composition, with a minimal effect on the lysine and arginine percentages in the insoluble form.

Characterization of a thermophilic L-rhamnose isomerase from Caldicellulosiruptor saccharolyticus ATCC 43494.

L-Rhamnose isomerase (EC 5.3.1.14, l-RhI) catalyzes the reversible aldose-ketose isomerization between L-rhamnose and L-rhamnulose. In this study, the L-rhi gene encoding L-RhI was PCR-cloned from Caldicellulosiruptor saccharolyticus ATCC 43494 and then expressed in Escherichia coli. A high yield of active L-RhI, 3010 U/g of wet cells, was obtained after 20 °C induction for 20 h. The enzyme was purified sequentially using heat treatment, nucleic acid precipitation, and ion-exchange chromatography. The purified L-RhI showed an apparent optimal pH of 7 and an optimal temperature at 90 °C. The enzyme was stable at pH values ranging from 4 to 11 and retained >90% activity after a 6 h incubation at 80 °C and pH 7-8. Compared with other previously characterized L-RhIs, the L-RhI from C. saccharolyticus ATCC 43494 has a good thermostability, the widest pH-stable range, and the highest catalytic efficiencies (k(cat)/K(M)) against L-rhamnose, L-lyxose, L-mannose, D-allose, and D-ribose, suggesting that this enzyme has the potential to be applied in rare sugar production.

Characterization of a salt-tolerant xylanase from Thermoanaerobacterium saccharolyticum NTOU1.

A xylanase gene was PCR-cloned from Thermoanaerobacterium saccharolyticum and expressed in Escherichia coli. The xylanase (XynA) consisted of a signal peptide, glycoside hydrolase family 10 domains, carbohydrate-binding modules, and surface layer homology domains. It was optimally active at 70-73°C and at pH 5-7. It had enhanced activity with NaCl with optimal activity at 0.4 M but was tolerant up to 2 M NaCl. The thermostable and salt-tolerant properties of this xylanase suggest that it may be useful for industrial applications.

Characterization of a thermophilic L-rhamnose isomerase from Thermoanaerobacterium saccharolyticum NTOU1.

L-rhamnose isomerase (EC 5.3.1.14, L-RhI) catalyzes the reversible aldose-ketose isomerization between L-rhamnose and L-rhamnulose. In this study, the L-Rhi gene encoding L-Rhi was PCR-cloned from Thermoanaerobacterium saccharolyticum NTOU1 and then expressed in Escherichia coli. A high yield of the active L-RhI, 9780 U/g of wet cells, was obtained in the presence of 0.2 mM IPTG induction. L-RhI was purified sequentially using heat treatment, nucleic acid precipitation, and anion-exchange chromatography. The purified L-RhI showed an apparent optimal pH of 7 and an optimal temperature at 75 °C. The enzyme was stable at pH values ranging from 5 to 9, and the activity was fully retained after a 2 h incubation at 40-70 °C. L-RhI from T. saccharolyticum NTOU1 is the most thermostable L-RhI to date, and it has a high specific activity (163 U/mg) and an acceptable purity after heat treatment, suggesting that this enzyme has the potential to be used in rare sugar production.

Simultaneous mutations up to six distal sites using a phosphorylation-free and ligase-free polymerase chain reaction-based mutagenesis.

In this study, we present an efficient phosphorylation-free and ligase-free PCR-based multiple site-directed mutagenesis that allows simultaneous mutations up to six distal sites. This method could be extended to any plasmid DNA that is isolated from dam(+)Escherichia coli strains, and the results showed that the simultaneously mutagenic efficiencies of quadruple mutation and sextuple mutation were up to 80% and 40%, respectively.

Identification of the essential catalytic residues and selectivity-related residues of maltooligosyltrehalose trehalohydrolase from the thermophilic archaeon Sulfolobus solfataricus ATCC 35092.

Maltooligosyltrehalose trehalohydrolase (MTHase) catalyzes the release of trehalose by cleaving the alpha-1,4-glucosidic linkage next to the alpha-1,1-linked terminal disaccharide of maltooligosyltrehalose. Mutations at residues D255, E286, and D380 were constructed to identify the essential catalytic residues of MTHase, while mutations at residues W218, A259, Y328, F355, and R356 were constructed to identify selectivity-related residues of the enzyme. The specific activities of the purified D255A, E286A, and D380A MTHases were only 0.15, 0.09 and 0.01%, respectively, of that of wild-type MTHase, suggesting that these three residues are essential catalytic residues. Compared with wild-type MTHase, A259S, Y328F, F355Y, and R356K MTHases had increased selectivity ratios, which were defined as the ratios of the catalytic efficiencies for glucose formation to those for trehalose formation in the hydrolysis of maltooligosaccharides and maltooligosyltrehaloses, respectively, while W218A and W218F MTHases had decreased selectivity ratios. When starch digestion was carried out at 75 degrees C and wild-type and mutant MTHases were, respectively, used with isoamylase and maltooligosyltrehalose synthase (MTSase), the ratios of initial rates of glucose formation to those of trehalose formation were inversely correlated to the peak trehalose yields.

A novel megaprimed and ligase-free, PCR-based, site-directed mutagenesis method.

In this study, we report a novel megaprimed and ligase-free, PCR-based, site-directed mutagenesis method modified from the QuikChange site-directed mutagenesis (QCM). One mutagenic oligonucleotide and one universal flanking primer were used to produce the complementary megaprimers that were then used to amplify the whole plasmid template. This method yields a mutagenesis efficiency ( approximately 90%) similar to that of QCM but uses only one mutagenic oligonucleotide instead of two of them, and the length of the oligonucleotide could be shorter. This method can be further extended to double mutations that are located at distant sites by using two mutagenic oligonucleotides and even to site saturation mutagenesis by introducing randomized codons.

Trehalose enhances transgene expression mediated by DNA-PEI complexes.

Using enhancers to improve the transfection efficiency of polyethylenimine (PEI) can circumvent the needs of chemical modifications as well as subsequent purification and characterization of the modified PEI. In this study, we found that incorporating trehalose into the transfection reagent could improve the transgene expression mediated by DNA-PEI complexes. Such enhancements were not observed when trehalose was replaced by other disaccharides. In an effort to explore the mechanisms, we examined how the timing of trehalose treatments and the durations of trehalose affected the percentages of cells expressing green fluorescent protein and the levels of intracellular ethidium monoazide labeled plasmid. Treatments with trehalose for 5-120 min prior to transfection could cause drops in transfection efficiency by 30-50%; such treatments, however, hardly affected the amounts of intracellular plasmid, indicating that the preexistence of intracellular trehalose could reduce transfection efficiency without lowering the endocytic activity. The transfection efficiency remained almost unchanged when the transfected cells were treated with trehalose after the removal of transfection reagents, indicating that trehalose had minimal effects on the machinery of protein synthesis. Despite the enhanced transgene expression, the presence of trehalose during transfection showed inhibitory effects on the internalization of DNA-PEI complexes. Additionally, the extent of enhancement in transgene expression strongly depended on the duration of trehalose. As the above observations suggested, only during the transfection process when complexes and trehalose coexisted, trehalose became an effective enhancer of transgene expression mediated by DNA-PEI complexes possibly by affecting the mechanisms of intracellular trafficking.

Using disaccharides to enhance in vitro and in vivo transgene expression mediated by a lipid-based gene delivery system.

BACKGROUND: Lipid-based vectors have been widely applied to in vivo and in vitro gene delivery. Disaccharides can effectively stabilize lipid membranes. This study examined whether disaccharides could enhance the transgene expression mediated by lipid-based vectors. METHODS: Different disaccharides were incorporated into the vectors prepared with DOTAP/protamine/DNA (LPD) or with DNA/cationic liposomes containing DOTAP, DOTAP/Chol, DOTAP/DOPE, or DC-Chol/DOPE. The levels of transgene expression and internalized plasmid of CHO cells were represented by the percentages of GFP-positive cells and the fluorescence intensity of ethidium-monoazide covalently labeled plasmid, respectively. The vectors containing either cellobiose or trehalose were also intravenously injected into mouse tail vein to investigate the potentials of in vivo applications. RESULTS: For enhancing the transgene expression, cellobiose was found to be effective for all the vectors whereas maltose decreased the effectiveness of DOTAP/Chol liposomes and LPD. For the internalization of plasmid, most disaccharides were able to increase the cellular delivery of DOTAP, DOTAP/Chol, and DOTAP/DOPE liposomes, but caused decreases in the cellular entry of DC-Chol/DOPE liposomes. An approximately linear correlation between the internalized plasmid and the transgene expression was observed for all the treatments in this study. When the vectors were administered to mouse by intravenous injection, 10-fold and 3-fold increases in the luciferase expression of lung were observed for DOTAP liposomes containing 330 mM cellobiose and trehalose, respectively. CONCLUSIONS: This study showed that using trehalose and cellobiose with a lipid-based delivery system provides a straightforward approach to effectively enhance both in vitro and in vivo transgene expression.

Protein engineering of Sulfolobus solfataricus maltooligosyltrehalose synthase to alter its selectivity.

Maltooligosyltrehalose synthase (MTSase) is one of the key enzymes involved in trehalose production from starch and catalyzes an intramolecular transglycosylation reaction by converting the alpha-1,4- to alpha,alpha-1,1-glucosidic linkage. Mutations at residues F206, F207, and F405 were constructed to change the selectivity of the enzyme because the changes in selectivity could reduce the side hydrolysis reaction of releasing glucose and thus increase trehalose production from starch. As compared with wild-type MTSase, F405Y and F405M MTSases had decreased ratios of the initial rate of glucose formation to that of trehalose formation in starch digestion at 75 degrees C when wild-type and mutant MTSases were, respectively, used with isoamylase and maltooligosyltrehalose trehalohydrolase (MTHase). The highest trehalose yield from starch digestion was by the mutant MTSase having the lowest initial rate of glucose formation to trehalose formation, and this predicted high trehalose yield better than the ratio of catalytic efficiency for hydrolysis to that for transglycosylation.